Abstract
The success of allotransplants is critically dependent on both tissue viability and efficient removal of potentially toxic cryopreservants. In this study we analysed the dimethyl sulphoxide (Me2SO) content of cardiovascular tissue samples stored in tissue banks and optimized a washing protocol to be used before surgical implant. We compared the Me2SO content of heart valves, arteries and veins and quantitatively determined by HPLC the washing kinetics of each group of tissue samples under strictly controlled conditions using an industrial washing medium(BASE).Our results showed that heart valves and arteries have significantly slower Me2SO release kinetics than veins. Approximately 20 % of the initial content of cryopreservant could still be detected in the valves and arterial tissue after 15 min
24 of continuous washing. Conversely, veins were almost completely cleared of the cryoprotectant under the same conditions. We propose a washing protocol consisting of two sequential washing with BASE for a total of 25 min for valves and arteries and 15 min for veins. In our hands, this protocol reliably ensures the removal of more than 95 % of the initial Me2SO content.

Abstract: The presence of residual antibiotics in tissue allografts after decontamination with antibiotic cocktails may result in widely documented adverse effects in predisposed subjects.

Moreover, antibiotic residues may mask contaminating microorganisms, resulting in falsely negative sterility tests, with potential risk of post-surgical infections. The objective of the present study was to define a rinsing procedure capable of eliminating antibiotic residues from cardiovascular, bone and skin tissues after decontamination with BASE.128. Different washing patterns, employing BASE medium, were applied.

The presence of antibiotic residues in tissue homogenates was assessed by agar diffusion test at different stages of tissue processing. To test whether antibiotic residues can result in falsely negative microbiological analysis, we induced a superficial tissue contamination with known inoculum concentration. By employing four different porcine tissues, we here report direct evidence that the presence of even limited amounts of antibiotics in decontaminated tissues interferes with sterility testing. This has implications in terms of increased risk of infections in allograft recipients.

To minimize this risk, we developed a procedure for extensive removal of antibiotics from allografts, allowing for subsequent detection of microbial contaminations that may occur during transportation, storage or processing prior to allograft transplantation. Our study emphasizes the importance of validating all processes and analytical methods in tissue banking, in order to warrant tissue safety.

This will minimize the risks of post-surgical infections as well as antibiotic-induced anaphylaxis in predisposed patients.

Abstract: Skin decontamination and cryopreservation can result in the presence of antibiotic and glycerol residues, which shall be removed before transplantation  to ensure tissue safety. Our study showed that the elimination of antibiotic residues from cryopreserved skin depends on the type of antibiotic, thus suggesting that specific rinsing procedures shall be established and validated for each antibiotic solution used to decontaminate tissue.

Abstract: We studied the optimal conditions to decontaminate and rinse cardiovascular tissues intended for transplantation with the aim of decontaminating effectively the tissue and minimizing the amount of residual antibiotics.

BASE.128 removed all contaminants from the tissue without affecting its integrity. In addition, decontamination with BASE.128 at 37°C for 6h followed by an overnight rinsing with BASE eliminated antibiotic residues, thus minimizing transmission to recipient and interference with microbiology tests.

Abstract: Elimination of Me2SO from cryopreserved allografts is one of the crucial steps in tissue processing phases in order to guarantee the tissue safety and quality. The aim of the study was to monitor the Me2SO removal from cryopreserved pig skin and heart valves and validate a rinsing procedure with BASE to eliminate Me2SO.

Abstract: Decontamination of tissue allografts in antibiotic cocktails can lead to the antibiotic carry-over effect, which in turn can result in the false negative microbiological analysis and in uncontrolled introduction of antibiotics in receiving patient.

Purpose: Elimination of the antibiotics carry-over through neutralization with resin mixture, designed specifically for elimination of antibiotics from tissues previously decontaminated with the industrially manufactured medical device BASE.128.

Methods: Sterile Amberlite resins (XAD16, XAD18, XAD4, PFC3500, CG50) and C18 resins were tested. Porcine heart valve, bone and skin segments were decontaminated at 37°C for 6h and at 4°C for 24h with BASE.128 containing Vancomycin, Gentamicin, Amphotericin B and Cefotaxim. Tissue samples were then incubated for 1h at room temperature under continuous stirring in presence of resin mixture. Removal of antibiotics from the broth and tissue homogenate was assessed by agar diffusion test. 10-100 cfu/ml of Pharmacopea reference microorganisms S. Aureus (SA), C. Albicans(CA), P. Aeruginosa (PA), and an additional bacterial strain: methicillin-resistant S. Epidermidis (SEMR) were used to test the compatibility of resins with microbial survival. Adhesion of microorganisms was evaluated by plating the medium on a Petri dish and quantifying bacteria before and after neutralization with different resins.

Results: Tissue samples introduced into the growth broth immediately after decontamination and without rinsing induced inhibition of bacterial growth of some tested microorganisms. One of the tested mixtures of non ionic hydrophobic adsorbent resins and exchange resins contrasted efficiently such carryover effect. The absence of inhibition areas in the agar diffusion test demonstrated the antibiotic removal from decontaminated tissue samples. The efficiency of the antibiotic removal from the tissue samples depended on antibiotic, time of contact between resin and antibiotic solution, quantity of resins and tissue. No interference with bacterial survival was present for all resins. However, the investigated ion exchange resins showed a small bacteriostatic effect against SA, SEM, PA but not CA.

Conclusions: Our study permitted to design an antibiotic-neutralizing mixture compatible with bacterial survival and proliferation. Neutralizing resins added directly in growth medium eliminated efficiently antibiotic carryover effect induced by not rinsed decontaminated tissues samples.

Abstract: Elimination of DMSO from cryopreserved skin allografts is one of the crucial steps in tissue processing in order to warrant tissue safety and quality. The aim of the study was to monitor the DMSO removal from cryopreserved human skin and validate the rinsing procedure of the tissue with the medical device BASE.

Abstract: Elimination of DMSO from cryopreserved allografts is one of the crucial steps in tissue processing in order to warrant tissue safety and quality. The aim of the study was to evaluate the DMSO removal from cryopreserved human
cardiovascular tissue after rinsing with the BASE medical device and to validate the rinsing procedure of the tissue with BASE.

Abstract: We evaluated the performances of the HTP medical devices in comparison with solutions currently used by the Cardiovascular Tissue Bank of Emilia-Romagna (CTBER) and the Cardiovascular Tissue Bank of Lombardy (CTBL).

Heart valves and blood vessels were procured from HB and NHB donors. Tissues were divided in half and processed in parallel using the HTP products or solutions prepared by the banks.